The synthesis of allolactose from amygdalin

Article abstract of DOI:10.1081/CAR-120023469

An original synthetic approach to the disaccharide allolactose (1) starting from the natural glycoside amygdalin (2) has been developed. The disaccharidic gentiobiose unit present in 2 has been transformed into the allolactose moiety using a four step pro

Full text of DOI:10.1081/CAR-120023469

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The Synthesis of Allolactose from Amygdalin
Davide Prosperi a d , Luigi Panza a , Dietmar Haltrich b , Micol Nonini c & Sergio Riva c
a Dipartimento di Scienze Chimiche , Alimentari, Farmaceutiche e Farmacologiche ,
Università del Piemonte Orientale , Via Ferrucci 33, 28100, Novara, Italy
b Institute of Food Technology , University of Agricultural Sciences (Boku) , Muthgasse 18,
1190, Vienna, Austria
c Istituto di Chimica del Riconoscimento Molecolare , C.N.R. , Via Mario Bianco 9, 20131,
Milano, Italy
d Istituto di Scienze e Technologie Molecolari , Consiglio Nazionale delle Ricerche , Via Golgi
19, 20133, Milano, Italy
Published online: 16 Aug 2006.
To cite this article: Davide Prosperi , Luigi Panza , Dietmar Haltrich , Micol Nonini & Sergio Riva (2003) The Synthesis of
Allolactose from Amygdalin, Journal of Carbohydrate Chemistry, 22:5, 267-274, DOI: 10.1081/CAR-120023469
To link to this article: http://dx.doi.org/10.1081/CAR-120023469
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, No. 5, pp. 267–274, 2003
The Synthesis of Allolactose from Amygdalin
Davide Prosperi,1,# Luigi Panza,1 Dietmar Haltrich,2
Micol Nonini,3 and Sergio Riva3,
*
1Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche,
Universita` del Piemonte Orientale, Novara, Italy
2Institute of Food Technology, University of Agricultural Sciences (Boku),
Vienna, Austria
3Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Milano, Italy
ABSTRACT
An original synthetic approach to the disaccharide allolactose (1) starting from the
natural glycoside amygdalin (2) has been developed. The disaccharidic gentiobiose
unit present in 2 has been transformed into the allolactose moiety using a four step
protocol based on the selective inversion of the C-4’ OH. The efficient removal of the
natural benzylic aglycone (a mandelonitrile moiety) was accomplished by catalytic
transfer hydrogenolysis. The behavior of allolactose with different enzymes is
also described.
Key Words: Allolactose; Amygdalin; Oxidoreductases; Analytical standard.
#Current address: Istituto di Scienze e Technologie Molecolari, C.N.R. Milano, Italy.
*
Correspondence: Sergio Riva, Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via
Mario Bianco 9, 20131, Milano, Italy; E-mail: sergio.riva@icrm.cnr.it.
267
DOI: 10.1081/CAR-120023469
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

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Prosperi et al.
INTRODUCTION
Allolactose (b-D-galactopyranosyl-(1!6)-a,b-D-glucopyranose, 1) is a reducing
disaccharide playing an important biological role in bacterial metabolism. For instance,
in E. coli allolactose regulates the activity of the so-called ‘‘lac-operon’’,[1,2] which
consists of three structural genes responsible for the uptake and hydrolysis of lactose
used as a carbon source. In the absence of lactose a repressor protein is bound to the
operator region of the lac operon, thus preventing RNA polymerase from binding to the
DNA and initiating the transcription. In the presence of lactose, a b-galactosidase
converts some lactose to allolactose (1), which binds to the tetramer repressor and
induces an allosteric change of this protein. As a consequence, the repressor protein is
no longer able to bind to DNA, so it falls off exposing the operator and its promoter
sequence for transcription.
Despite its biological importance and its simple structure, allolactose is presently
not commercially available. Synthetic approaches to this molecule exploited either the
usual protocols of chemical condensation between suitable galactose donors and
glucose acceptors by Koenigs-Knorr glycosylation,[2] or – mimicking nature – a b-
galactosidase catalyzed trans-glycosylation.[3,4] The latter approach led to the formation
of a mixture of mono-, di- and trisaccharides that had to be carefully separated.
In the framework of an EEC-project pursuing enzymatic lactose valorization, we
became interested in producing allolactose to be used as a chromatographic standard
and as a potential substrate for newly isolated sugar-oxidoreductases. The use of natural
building blocks can have some advantages for the synthesis of di- and oligosacchar-
ides,[5,6] as already well-documented in the literature. Accordingly, we took into
consideration the elaboration of a natural disaccharide derivative as a starting material,
in order to avoid chemical or enzymatic glycosylation. The synthetic protocol that has
been developed as well as the performances of allolactose with different enzymes are
described in this report.
RESULTS AND DISCUSSION
Amygdalin (2) is a commercially available natural cyanogenic glycoside which
occurs in the seeds of Rosaceae, principally in bitter almonds.[7] Its chemical structure
appeared quite suitable for our purposes in that the disaccharidic unit might be
chemically elaborated from the actual gentiobiose (b-D-glucopyranosyl-(1!6)-D-
glucose) moiety to the desired allolactose (b-D-galactopyranosyl-(1!6)-D-glucose)
target by selective inversion of the C-4’ OH followed by the removal of the natural
benzylic aglycone (a mandelonitrile moiety) by catalytic hydrogenolysis.
Scheme 1 shows the optimized protocol that has been developed. The new
compound 4 was prepared from amygdalin by a two-step standard procedure and its
4,6-O-benzylidene acetal was efficiently and selectively reduced following the protocol
suggested by DeNinno et al. (triethylsilane – trifluoroacetic acid – trifluoroacetic
anhydride)[8] to give the previously undescribed monohydroxy derivative 5 (the three
synthetic steps gave 5 in 59% overall isolated yields from 2) After different attempts,
the inversion of the equatorial 4’-OH to the desired axial hydroxyl was accomplished
via the triflate intermediate 5a (not isolated), exploiting an approach already used for

Synthesis of Allolactose
269
Scheme 1. Synthesis of allolactose from amygdalin. a) PhCH(OMe)2, H+, 35°C; b) Ac2O, DMAP,
Et3N; c) Et3SiH, Trifluroacetic anhydride, TFA; d) Tf2O, –15°C; then H2O, reflux; e)
Cyclohexadiene, Pd/C, reflux; f) IRA-400 resin (OHꢀ form).
the inversion of C-4 OH of glucosamine.[9] This compound was prepared by reacting 5
with triflic anhydride in CH2Cl2; water was added to the reaction mixture which was
then boiled to give 6 (inversion of the 4’-OH was accompanied by the migration of the
3’-OAc) as the main product in 67% isolated yields (5 was also recovered in 22% yield,
due to the undesired triflate hydrolysis).
The presence of the axial acetate at C-4’ and of the non-acetylated OH at C-3’ was
confirmed by NMR analysis. The 1H NMR spectrum of the precursor gentiobiosyl
derivative 5 showed five triplets between 4.85 and 5.11 ppm, due to H-3’, H-2’, H-4, H-
1
3 and H-2. Only four triplets were present in the same region of the H NMR spectrum
of 6 and, in addition, a newly appeared broad doublet (J=3.9 Hz), integrated as one
proton, resonated at 5.39 ppm, clearly due to the equatorial H-4’. A 2D-COSY
experiment showed that this signal was coupled to a broad triplet at 3.81 ppm and to a
doublet of doublet (J=8.6 Hz and J=3.9 Hz) at 3.90 ppm. The latter signal, additionally
coupled to a triplet at 5.0 ppm (H-2’), was significantly (more than 1 ppm) downfield
shifted upon acetylation (compound 6a) and it was therefore assigned to the axial H-3’,
while the broad triplet at 3.81 ppm was the signal due to H-5’.
The final deprotection of compound 6 was best achieved by catalytic transfer
hydrogenation (substrate boiled in EtOH in the presence of 1,4-hexadiene and Pd/C)[10]

270
Prosperi et al.
to remove the benzylic substituents (the mandelonitrile aglycone and the C-6’ benzyl
group) to give the intermediate 7, followed by methanolysis of the acetate groups in the
presence of an anion exchange resin (OHꢀ). Both the reactions were quantitative and
pure 1 was recovered by solvent evaporation.
The structure of 1 was proved to be allolactose by 13C NMR. The presence of a
reducing disaccharide was confirmed by the three anomeric signals at 106.2 (C-1’), 98.9
(C-1b) and 95.0 (C-1a) ppm. Additionally, a diagnostic upfield-shifted signal at 71.5
ppm, due to the C-4’ of a galactose moiety, was observed. All these data were in full
agreement with a literature report.[11]
Allolactose was then tested for different applications. Initially, it was a useful
standard for the analysis and the identification (by capillary electrophoresis) of a
mixture of oligosaccharides produced by transglycosylation of lactose with thermo-
stable b-glycosidases.[12] Secondly, 1 was used for investigating the substrate spe-
cificity of disaccharide-converting enzymes. The first biocatalyst considered was a
cellobiose dehydrogenase, an enzyme that is produced extracellularly by a number of
wood- and cellulose-degrading fungi when grown on cellulose, and that, in our case,
was isolated from the plant pathogen Sclerotium (Athelia) rolfsii.[13] These biocatalysts
oxidize the reducing end of cellobiose and cello-oligosacharides to their corresponding
1,5-lactones, which are subsequently hydrolyzed to the corresponding carboxylic acids
in aqueous environments. Knowledge of the specificity of these enzymes is important
since they might be useful for the specific determination of lactose, e.g., in samples of
oligosaccharides produced with the above described process,[12] in view of lactose
intolerance of consumers that might use these oligosaccharide mixtures. It was found
that the cellobiose dehydrogenase from Sclerotium (Athelia) rolfsii is indeed very
specific for reducing disaccharides carrying a b-1,4-glycosidic linkage: cellobiose and
lactose were converted to the corresponding bionic acid, while neither allolactose nor
other b-1,6- (gentiobiose), b-1,2- (sophorose) and b-1,3 positional isomers of cellobiose
and lactose were oxidized.[13] Based on these results the application of cellobiose
dehydrogenase for the analytic purpose outlined should be feasible. Finally, allolactose
was regioselectively oxidized at C-2 OH by a pyranose 2-oxidase isolated from the
basidiomycete Trametes multicolor.[14] This enzyme catalyzes the oxidation of several
aldopyranoses at the C-2 position to yield the corresponding aldos-2-uloses (osones).
As shown in Scheme 2, this was the first step of an enzymatic isomerization of a series
of aldose-disaccharides into the corresponding ketoses, a transformation that cannot be
Scheme 2. Isomerization of aldose-disaccharides into the corresponding ketones by an
oxidoreductive route employing pyranose oxidase and aldose reductase (see Ref. [14]).

Synthesis of Allolactose
271
catalyzed by the available glucose (xylose) isomerases, enzymes that are used for the
conversion of D-glucose into D-fructose. Four 1,6-linked disaccharides (allolactose and
the commercially available gentiobiose, isomaltose [a-D-Glcp-(1!6)-D-Glc] and
melibiose [a-D-Galp-(1!6)-D-Glc]), were converted into the corresponding glycosyl
D-arabino-hexos-2-uloses. Gentiobiose and allolactose were clearly the preferred sub-
strates showing, after 2 hours, a 90 and 72% conversion, respectively, while melibiose
and isomaltose required much longer reaction times.[14]
In conclusion, we have reported an original synthetic approach to the di-
saccharide allolactose (which avoids the use of heavy metal salts, as in the Koenigs-
Knorr glycosylation, or tedious chromatographic separation, as in the glycosidases-
catalyzed transglycosylation) and we have summarized the performances of different
enzymes with this disaccharide.[13,14] The facile removal of the mandelonitrile
aglycone (that might be considered as a ‘‘natural’’ anomeric protective group) and the
easy access to partly protected intermediates, such as 5 and 6, which might be useful
for the synthesis of more complex derivatives, make amygdalin an interesting and
available building block for oligosaccharides carrying a reducing gentiobiose or
allolactose moiety.
EXPERIMENTAL
General methods. Amygdalin (2) and the other reagents were obtained from
1
Sigma-Aldrich. H NMR and 13C NMR spectra were recorded on a Bruker AC-300
instrument. [a]D were measured using a Perkin-Elmer 141 polarimeter. Melting points
were determined using a Kofler apparatus. TLC was performed on Merck Silica Gel 60
F254 plates.
4’,6’-O-Benzylidene amygdalin (3). Amygdalin 2 (4 g, 9 mmol) was dissolved
in dry dimethylformamide (20 mL). Benzaldehyde dimethyl acetal (3.3 mL, 22 mmol)
and a catalytic amount of p-toluenesulfonic acid hydrate (50 mg) were added to the
solution in a round-bottomed flask, which was then attached to a rotary rotavapor
immersed in a water bath at 35°C. The reaction was completed in 4 hours (TLC:
AcOEt/MeOH/H2O, 80:20:5 v/v). Et3N (1 mL) was added and the solvent was then
evaporated at reduced pressure. The solid obtained after evaporation was dissolved in
hot methanol (10 mL) (if necessary, some drops of water could be added) and allowed
to precipitate overnight, giving 3 as a white solid. Yield 4.35 g (91%); mp 232–234°C;
1
[a]D20 = –68.0° (c 0.50, DMSO); H NMR (D6-DMSO+D2O): d 7.7–7.2 (m, 10 H,
Ar), 5.98 (s, 1 H, CHCN), 5.61 (s, 1 H, PhCH), 4.66 and 4.38 (2 d, 1 H each, J 8.5 Hz
and J 8.0, H-1 and H-1’), 4.25 (dd, 1 H, J 10.5 Hz, J 4.0 Hz, H-6’eq), 4.09 (d, 1 H, J
11.5 Hz, H-6a), 3.8–3.1 (several m, 10 H).
Anal. Calcd for C27H31NO11: C, 59.45; H, 5.69; N, 2.57. Found: C, 59.57; H, 5.64;
N, 2.62.
2,3,4,2’,3’-Penta-O-acetyl-4’,6’-O-benzylidene amygdalin (4). Compound 3
(4.25 g, 8 mmol) was dissolved in 100 mL of CH2Cl2. Dimethylaminopyridine (150
mg) and Et3N (11 mL, 79 mmol) were added and the mixture was cooled at 0°C.
Acetic anhydride (15 mL, 158 mmol) was added dropwise at 0°C. When the addition

272
Prosperi et al.
was terminated, the mixture was allowed to react for 2 h at room temperature (TLC:
AcOEt/hexane 4:6). The mixture was washed with H2O (80 mL), 5% w/v NaHCO3 (80
mL) and H2O again (80 mL), until the pH was neutral. The organic solution was
separated from the aqueous phase, dried with Na2SO4 and concentrated at reduced
pressure, providing the product 4 as a white solid (5.2 g, 88% yield): mp 183–184°C;
1
[a]D20 = –66.2° (c 0.50, CHCl3,); H NMR (CDCl3): d 7.5–7.3 (m, 10 H, ArH), 5.56
and 5.49 (2 s, 1 H each, CHCN and PhCH), 5.33 (t, 1 H, J 9,8 Hz) and 5.14–4.89 (m,
4 H) : H-2, H-3, H-4, H-2’, H-3’, 4.69 and 4.46 (2 d, 1 H each, J 7.9 Hz, H-1 and H-1’),
4.37 (dd, 1 H, J 10.0 Hz, J 4.4 Hz, H-6’eq), 3.92–3.49 (several m, 6 H), 2.10, 2.04,
1.99, 1.97 (4 s, 15 H total, Ac).
Anal. Calcd for C37H41NO16: C, 58.81; H, 5.43; N, 1.85. Found: C, 58.71; H, 5.54;
N, 1.88.
2,3,4,2’,3’-Penta-O-acetyl-6’-O-benzyl amygdalin (5). Carefully dried compound
4 (5.2 g, 7 mmol) was dissolved in dry CH2Cl2 (30 mL) in an inert atmosphere at 0°C.
Triethylsilane (5.7 mL, 36 mmol), trifluoroacetic anhydride (3.0 mL, 21 mmol) and
trifluoroacetic acid (2.7 mL, 36 mmol) were added dropwise. After stirring at room
temperature for 1 h (TLC: AcOEt/hexane 1:1), the solution was diluted with CH2Cl2
(100 mL) and washed successively with H2O (60 mL), 5% NaHCO3 (60 mL) and H2O
again (20 mL). The organic layer was dried with Na2SO4 and the solvent was
evaporated. The product was purified by flash chromatography (AcOEt/hexane 1:1),
furnishing 5 as a white solid in 74% yield (3.85 g): mp168–169°C; [a]D20 = –47.1° (c
1
0.50, CHCl3,). H NMR (CDCl3): d 7.5–7.2 (m, 10 H, ArH), 5.53 (s, 1H, CHCN),
5.12–4.85 (m, 5 H, H-2, H-3, H-4, H-2’, H-3’), 4.61 and 4.54 (2 d, 1 H each, J 12.2
Hz, PhCH2), 4.58 and 4.39 (2 d, 1 H each, J 8.0 Hz, H-1 and H-1’), 3.89–3.51 (several
m, 8 H), 2.10, 2.09, 1.99, 1.98 (4 s, 15 H total, Ac); 13C NMR (CDCl3): d 171.3 (CO),
170.1 (CO), 169.8 (CO), 169.4 (CO), 168.9 (s, CO), 137.7, 132.1, 130.4, 129.2, 128.4,
127.7, 117.1 (CN), 100.5 and 97.6 ( C-1 and C1’), 75.4, 74.2, 73.7 (PhCH2), 73.6, 72.5,
71.3, 70.9, 70.4, 69.9, 68.7, 67.8, 67.6, 20.8, 20.4.
Anal. Calcd for C37H43NO16: C, 58.65; H, 5.68; N, 1.85. Found: C, 58.64; H, 5.74;
N, 1.86.
Phenyl cyanomethyl 2’,4’-di-O-acetyl-6’-O-benzyl- -D-galactopyranosyl-(1?6)-
(2,3,4-tri-O-acetyl- -D-glucopyranoside) (6). Compound 5 (300 mg, 0.40 mmol) was
dissolved in dry CH2Cl2 (6 mL) under an inert atmosphere. Pyridine (200 mL, 2.48
mmol) was added and the mixture was cooled at ꢀ15°C. Triflic anhydride (180 mL,
1.07 mmol) was added dropwise and the formation of the triflate 5a was observed in
TLC (eluent AcOEt/hexane 1:1). When the reaction was complete (1 h), H2O was
added (250 mL) and the resulting solution was refluxed for 4 h. Subsequently, the
mixture was diluted with CH2Cl2 (20 mL), washed with 5% HCl (10 mL), NaHCO3 (20
mL) and H2O (20 mL). The organic solution was dried with Na2SO4 and concentrated
at reduced pressure. The product was purified by flash chromatography (AcOEt/hexane
1:1), furnishing 6 as a white solid in 67% yield (200 mg) together with 66 mg of
starting compound 5. Compound 6: mp 165–166°C; [a]D20 = –40.5° (c 0.41, CHCl3);
1H NMR (CDCl3, COSY): d 7.5–7.2 (m, 10 H, ArH), 5.54 (s, 1 H, CHCN), 5.38 (br d,
1 H, J 3.8 Hz, H-4’), 5.12–4.97 (m, 3 H, H-2, H-3, H-2’), 4.91 (br t, 1 H, J 8.8 Hz, H-4),

Synthesis of Allolactose
273
4.55 and 4.41 (2 d, 1 H each, J 8.0 Hz, H-1 and H-1’), 4.53 and 4.44 (2 d, 1 H each, J
11.6 Hz, PhCH2), 3.95–3.85 (m, 2 H, H-3’ and H-6a), 3.81 (br t, 1H, J 6.5 Hz, H-5’),
3.68–3.50 (m, 4H, H-5, H-6b, H-6’a, H-6’b), 2.15, 2.09, 1.96, 1.95, 1.93 (5 s, 15 H
total, Ac). 13C NMR (CDCl3): d 171.1 (CO), 171.0 (CO), 170.2 (CO), 169.5 (CO),
169.0 (CO), 132.2, 130.4, 129.3, 128.5, 127.9, 127.8, 117.1 (CN), 100.9 and 97.9 (C-1
and C1’), 73.7, 73.6, (PhCH2), 72.6, 72.5, 71.0, 70.2, 68.8, 68.0 (C-6 and C-6’), 67.8
(d), 21.1, 20.8, 20.5;
Anal. Calcd for C37H43NO16: C, 58.65; H, 5.68; N, 1.85. Found: C, 58.68; H, 5.71;
N, 1.85.
Conventional acetylation gave phenyl cyanomethyl 2’,3’,4’-di-O-acetyl-6’-O-
benzyl- -D-galactopyranosyl-(1?6)-2,3,4-tri-O-acetyl- -D-glucopyranoside (6a) as
an oil. 1H NMR (CDCl3, COSY) : d 7.5–7.2 (m, 10 H, ArH), 5.59 (s, 1 H, CHCN),
5.48 (br d, 1 H, J 3.6 Hz, H-4’), 5.21 (dd, 1 H, J 9.4 Hz, J 7.8 Hz, H-2’), 5.11–4.96
(m, 3 H, H-2, H-3, H-3’), 4.90 (br t, 1 H, J 8.8 Hz, H-4), 4.55 (d,1 H, J 7.8 Hz, H-1’),
4.53 and 4.41 (2 d, 1 H each, J 11.9 Hz, PhCH2), 4.36 (d, 1H, J 8.0 Hz, H-1), 3.95–
3.84 (m, 2 H, H-5’ and H-6a), 3.68–3.45 (m, 4H, H-5, H-6b, H-6’a, H-6’b), 2.11, 2.07,
1.98, 1.95 (4 s, 18 H total, Ac).
Anal. Calcd for C39H45NO17: C, 58.57; H, 5.63; N, 1.75. Found: C, 58.65; H, 5.67;
N, 1.73.
Allolactose ( -D-galactopyranosyl-(1?6)- , -D-glucopyranose, 1). Compound
6 (50 mg, 0.066 mmol) was dissolved in absolute ethanol by refluxing under nitrogen
atmosphere. Pd/C (50 mg) and cyclohexadiene (300 mL, 3.171 mmol) were added and
the solution was heated at 90°C. After 30 min the phenyl cyanomethyl aglycone was
removed first (after approximately 30 min) and 8 h later the benzyl group was also
completely eliminated (TLC: AcOEt). The product was filtered over packed celite and
dried. The resulting solid intermediate compound 7 (37 mg, 0.066 mmol) was
dissolved in methanol (1 mL) and complete deacetylation was accomplished in 1 h by
adding an Amberlite IRA-400 resin (OH– form) (TLC: AcOEt/MeOH/H2O 8:3:1). The
resin was filtered and the solvent evaporated. The reaction proceeded quantitatively
providing 21 mg (0.061 mmol) of 1 as a pure white solid: mp 174–176°C (lit.[15]
174–176°C); [a]D20 = 52.1° (c 0.33, H2O) 5 min after soln (lit.[15] 54.2°), 32.0° after
1h, 26.3 after 24 h (lit.[15] 30.7°); 13C NMR (D2O): d 106.2 (C-1’), 98.9 (C-1b), 95.0
(C-1a), 78.6 (C-3b), 78.0 (C-5), 77.8 (C-5b), 76.9 (C-2b), 75.6 (C-3’ and C-3a), 74.3
(C-2a), 73.6 (C-2’), 73.4 (C-5a), 72.4 (C-4a and C-4b), 71.5 (C-4’), 71.6 and 71.4 (C-
6a and C-6b), 64.0 (C-6’); (Lit.:[11] 100 MHz, D2O: d 106.1 (C-1’), 98.8 (C-1b), 94.9
(C-1a), 78.5 (C-3b), 78.0 (C-5’); 77.7 (C-5b), 76.9 (C-2b), 75.5 (C-3’ and C-3a), 74.2
(C-2a), 73.6 (C-2’), 73.3 (C-5a), 72.3 (C-4a and C-4b), 71.5 (C-4’, C-6a and C-6b),
63.8 (C-6’).
Anal. Calcd for C12H22O11: C, 42.11; H, 6.43. Found: C, 42.12; H, 6.48.
ACKNOWLEDGMENT
This work was supported by an European Commission grant (FAIR CT96-1048).

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Prosperi et al.
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Received Novermber 7, 2002
Accepted April 9, 2003